Analysis of the α/β T-cell receptor repertoire by competitive and quantitative family-specific PCR with exogenous standards and high resolution fluorescence based CDR3 size imaging

The characterization of the human T-cell receptor (TCR) repertoire in various physiological and pathological conditions has become an important tool in studies of the immune response. Therefore, a number of PCR based strategies for the semiquantitative analysis of the TCR repertoire have been described. Family specific amplification of TCR cDNA has been employed in a number of studies often with contradictory results. We have developed a strategy utilizing exogenous standards with homologous primer binding sites for the quantitative analysis of the α/β T-cell receptor repertoire. This system allows the detection of even minute differences in T-cell populations based on quantitative PCR (Q-PCR) and competitive PCR (C-PCR). Results presented here demonstrate that expansions of T-cell subsets as defined by the specificity of the variable gene segments can be readily monitored when exceeding 1% of the total repertoire. In addition, the proposed method reveals direct information of CDR3 size heterogeneity and can be used to estimate the T-cell repertoire complexity and monitor clonal expansions. We discuss variables such as cell number and experimental conditions influencing accuracy and reproducibility of the analyses. We have used this protocol based on non-radioactive techniques for characterization of the fine specificity of the T-cell repertoire in peripheral and organ-infiltrating T-lymphocytes. The analyses revealed information about polyclonal or clonal expansion of T-cells in vivo and in vitro following various stimuli such as superantigenic stimulation of T-cell subsets as well as antigen-driven shaping of the α/β T-cell repertoire in autoimmune and infectious diseases.     

Hyperplastic foci in chronic liver disease: Their proliferative activity assessed by nucleolar organizing region

In the cirrhotic and precirrhotic liver, there may be small foci with increased cellularity and amphophilic cytoplasm. These are microscopic lesions that do not form macroscopically detectable nodules, which differ from the macroscopically apparent nodules of dysplastic nodules. In the present study, we assessed the proliferating activity of 12 hyperplastic foci in 11 patients with cirrhosis or chronic hepatitis, by staining for agyrophilic nucleolar organizing regions (AgNOR). The mean AgNOR count per nucleus in the hyperplastic foci ranged from 0.96 to 1.36 (mean, 1.13; SD 0.12), and from 0.81 to 1.06 (mean, 0.94; SD 0.08) in the controls. The AgNOR count In the hyperplastic foci was significantly higher than that In the controls (P> 30.01). Small hyperplastic foci show Increased proliferative activity. Further study on these foci is required to clarify their relation to hepatocarcinogenesis.     

Role of the 2 adenine (g.11293_11294insAA) insertion polymorphism in the 3' untranslated region of the factor VII (FVII) gene: molecular characterization of a patient with severe FVII deficiency

Polymorphic variants in the gene encoding factor VII (F7) affect the plasma levels of this coagulation protein and modify the clinical phenotype of FVII deficiency in some patients. In this study we report the in vitro functional analysis of a novel polymorphic variant located in the 3' untranslated region of F7: g.11293_11294insAA. To determine whether this variant regulates FVII expression, we initially compared an expression vector containing FVII cDNA with g.11293_11294insAA with the FVII wild-type (WT) construct. The kinetics of mRNA production showed that the insertion decreases the steady-state FVII mRNA levels. To assess whether the insertion influences the phenotype of FVII-deficient patients, we evaluated its effect on the expression of FVII in a patient with severe FVII deficiency (undetectable FVII activity and antigen) carrying two additional homozygous missense variations (p.Arg277Cys and p.Arg353Gln). The two substitutions alone reduced the expression of FVII activity and antigen in vitro, but with the insertion polymorphism in our expression vector the patient's phenotype of undetectable plasma FVII was recapitulated. The insertion polymorphism in the 3' untranslated region of F7 is another modifier of FVII expression that might explain the poor genotype-phenotype correlation in some FVII-deficient patients.     

Neuronal responses to horizontal neck deflection in the group X region of the cat's medullary brainstem

Summary Neuronal responses to natural stimulation of neck proprioceptors were studied in the region of the small cell group x in the dorsolateral medullary brainstem of slightly anesthetized and paralyzed cats. Stimulation consisted of horizontal trunk rotations about C₁ with the head fixed in space. Out of 74 neurons recorded, 92% showed an increase in discharge rate with ipsilateral neck stretch and a decrease with contralateral stretch (Type N I responses); 8% showed the reverse response pattern (Type N II responses). In the primary head-to-trunk position, almost all neurons had tonic activity that probably stemmed from prestretched neck proprioceptors. Responses to sinusoidal stimulation and position trapezoids showed a static (position-sensitive) as well as a dynamic (essentially velocitysensitive) component. The relative weight of the two components varied considerably among the neurons. It was not possible to distinguish discrete neuronal populations on the basis of the dynamic characteristics. There was no evidence of a convergent input from other receptor systems, such as the horizontal canal system. Several neurons responded to muscle tapping and showed an increase of the velocity component following systemic injection of succinylcholine. We take this as evidence that they may receive input from muscle spindle receptors.Supported by the Deutsche Forschungsgemeinschaft, SFB 70/U4     

颈静脉孔区的显微解剖及定位标志研究

目的:研究颈静脉孔区的显微外科解剖,探讨寰椎横突(TPA)、头侧直肌、二腹肌沟、颈静脉突等解剖标志在颈静脉孔区病变手术中的定位意义。方法:成人头颈标本15例,男13例,女2例,红色乳胶灌注颈总动脉和椎动脉。手术显微镜下(×3～×30)逐层显露颈静脉孔区结构,明确该区显微解剖特征、相关的解剖标志及其定位意义。结果:颈静脉孔区多数重要的解剖结构均可以TPA为参照标志予以明确。二腹肌后腹位于其浅层。TPA的后方为枕下三角,三角内有椎动脉、椎静脉丛和颈1神经通过。头侧直肌起始于TPA的外表面,止于枕骨颈静脉突的下表面,可作为确定颅外颈静脉孔的解剖标志。茎突位于TPA的前方,颈内静脉、迷走神经、副神经、舌下神经穿行于茎突与TPA之间,颈内动脉位于颈内静脉的前内侧。二腹肌、颈静脉突、颈动脉嵴对定位面神经、颈静脉孔及舌咽神经等结构具有重要意义。结论:颈静脉孔区解剖结构复杂,利用寰椎横突、头侧直肌、二腹肌沟、颈静脉突等解剖标志有助于明确此区域重要的解剖结构,避免术中不必要的损伤。     

经额入路行蝶鞍区肿瘤手术的应用解剖

目的　为经额入路蝶鞍区肿瘤切除术提供解剖学资料。方法　采用解剖技术对 35个甲醛固定 ,红色明胶动脉灌注的尸头标本蝶鞍区的有关结构进行观察。结果　两侧视神经管颅口内缘间距为 11.0 9± 2 .2 2mm ,视交叉前缘至交叉前沟前缘间距为 7.0 2± 1.86mm。垂体上面与鞍隔平面有三种位置关系 :垂体上面通过鞍隔孔露出鞍隔平面以上者 3例 ( 8.75% ) ,在鞍隔平面以下者 13例 ( 37.14% ) ,与鞍隔平面平齐者 19例 ( 54.2 9% )。两侧颈内动脉在穿经海绵窦上壁处间距为 13.2 5± 2 .4 8mm ,大脑前动脉发起处 ,两侧颈内动脉之间距为 17.86± 1.55mm。结论　经额入路蝶鞍区肿瘤切除术主要是通过交叉前间隙 ,在颈内动脉之间的区域操作 ,手术中要保护颈内动脉、视神经等结构 ,以减少并发症的发生     

充满型及非充满型胆囊结石粘膜病理形态及核仁组成区的研究

目的：探讨充满型及非充满型胆囊结石粘膜组织病理学的差异及相应外科治疗措施,方法：收集1995年10月至1996年10月期间的胆囊结石标本做常规病理染色及嗜银蛋白染色分析。结果：发现充满型及非充满型结石组胆囊粘膜不典型发生率及AgNOR数目均有显著差异。结论：一经诊断为充满型胆囊结石,特别是中老年肥胖妇女,应早期手术切除病变胆囊。     

完全神经内镜下经Poppen锁孔入路松果体区手术的应用解剖学研究

目的 解剖观察完全神经内镜下经Poppen锁孔入路开颅松果体区手术的相关解剖结构及其特征,并探讨该术式的可行性。方法 选取12具经10%甲醛固定、红蓝乳胶灌注的成人尸头湿标本进行实验观察,其中男7例、女5例,年龄34~71岁。应用随机数字表法将12具标本随机分为内镜组和显微组,每组6具,分别采用完全神经内镜Poppen锁孔入路和显微镜常规Poppen入路进行模拟开颅手术松果体区手术。模拟手术中,利用神经导航对松果体区以及手术间隙进行观察测量：（1）观察2组松果体区重要解剖结构;（2）内镜组术中,于剪开小脑幕前后,分别测量松果体区暴露面积,并采用配对t检验进行比较;（3）内镜组与显微镜组术中,分别测量第1、2、3手术间隙的暴露面积,并采用独立样本t检验进行组间比较;（4）其他重要解剖结构间距的神经内镜测量。结果 （1）2种入路均可观察到双侧基底静脉、小脑中脑裂静脉、大脑内静脉、大脑后动脉、小脑上动脉等重要血管,以及滑车神经、四叠体、胼胝体压部和松果体等重要解剖结构,但显微镜常规Poppen入路的手术通道狭窄、倾斜,视野局限。（2）内镜组模拟手术中,剪开小脑幕前后松果体区显露面积分别为（73.14±3.38）mm²和（127.77±7.90）mm²,剪开后明显大于剪开前,差异有统计学意义（t=28.84,P<0.001）。（3）内镜组和显微镜组模拟手术中,第1、2、3手术间隙的暴露面积分别为（20.93±2.49）mm²、（72.55±4.18）mm²、（208.57±11.79）mm²和（9.12±1.12）mm²、（53.45±3.17）mm²、（175.29±9.98）mm²,内镜组均大于显微镜组,差异均有统计学意义（t=14.92、12.61、7.41,P值均<0.001）。（4）神经内镜测量显示：双侧基底静脉最大距离为（14.41±0.94）mm,双侧小脑中脑裂静脉最大距离为（8.23±0.84）mm,双侧大脑内静脉最大距离为（8.41±0.96）mm,双侧大脑内静脉最小距离（第1间隙最窄长度）为（2.58±0.22）mm,松果体中心点至丘脑枕部中心点距离为（16.83±1.16）mm。结论 完全神经内镜下经Poppen锁孔入路模拟手术中间隙恒定,可安全到达松果体区;与显微镜常规Poppen入路相比,完全神经内镜Poppen锁孔入路的手术操作空间更大,松果体区显露得更充分。